Methods of treating tissue calcification

ABSTRACT

The present invention provides a method of treating NPP1 deficiency or NPP1-associated disease such as idiopathic infantile arterial calcification (IIAC), pseudoxanthoma elasticum, vascular calcification in chronic kidney disease (VCCKD), insulin resistance, hypophosphatemic rickets, myocardial ischemia, joint calcification, angioid streaks, and ossification of the posterior longitudinal ligament of the spine. The present invention provides a method for treating tissue calcification by administering soluble NPP1 to produce a transient increase in serum pyrophosphate levels.

RELATED APPLICATIONS

This application is a United States National Phase under 35 U.S.C. § 371of International Application No. PCT/US2015/066646, filed on Dec. 18,2015, which claims the benefit of U.S. Provisional Application62/094,943, filed on Dec. 19, 2014 and U.S. Provisional Application No.62/249,781, filed on Nov. 2, 2015. The entire teachings of the aboveapplications are incorporated herein by reference. This application is areissue of U.S. Pat. No. 10,493,135, issued Dec. 3, 2019, which issuedfrom U.S. patent application Ser. No. 15/536,880, filed Mar. 16, 2017,which is a United States National Phase under 35 U.S.C. § 371 ofInternational Application No. PCT/US2015/066646, filed on Dec. 18, 2015,which claims the benefit of U.S. Provisional Application No. 62/094,943,filed on Dec. 19, 2014 and U.S. Provisional Application No. 62/249,781,filed on Nov. 2, 2015. The entire teachings of the above applicationsare incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name: 081245-0208_ascii.txt; Size: 88,556 bytes; and Date ofCreation: Dec. 15, 2015) filed with the application is incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION

Vascular calcification can be characterized by formation of very small,dispersed crystals of hydroxyapatite (HA) and as large calcifieddeposits in vascular tissue, such as arteries. (Amann, K. Clin J Am SocNephrol 2008, 3, 1599-605). Extracellular pyrophosphate (PPi) is a keyendogenous inhibitor of vascular calcification by inhibiting HAformation. (Lomashvili, K. A. et al., J Am Soc Nephrol 2004, 15,1392-1401; Fleisch, H. et al., Nature 1966, 212, 901-903).

Ectonucleotide pyrophosphatase pyrophosphorylase (NPP1) is an ectoenzymethat cleaves ATP to produce extracellular pyrophosphate (PPi).Pyrophosphate is a potent inhibitor of hydroxyapatite formation and,under normal conditions, functions to inhibit vascular calcification.

Deficiency of NPP1 in humans results in reduced circulating PPi levelsand has been implicated in conditions such as arterial calcification andgeneralized arterial calcification of infancy (GACI). (Rutsch, F. etal., Am J Pathol 2001, 158, 543-554). When fed a high-phosphate diet,mice lacking NPP1 (Enpp1^(−/−)) also have reduced PPi levels and exhibita similar phenotype as NPP1 deficient humans. (Harmey, D. et al., Am JPathol 2004, 164, 1199-1209). Vascular calcification is also awell-recognized and common complication in chronic kidney disease (CKD)and end-stage renal disease (ESRD) subjects, and is associated withincreased morbidity and mortality. (Giachelli, C. J Am Soc Nephrol 2004,15, 2959-64; Raggi, P. et al., J Am Coll Cardiol 2002, 39, 695-701).

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1/ENPP1/PC-1)deficiency is a rare disease caused by mutations in NPP1, a type IItransmembrane glycoprotein. NPP1 cleaves a variety of substrates,including phosphodiester bonds of nucleotides and nucleotide sugars andpyrophosphate bonds of nucleotides and nucleotide sugars. NPP1deficiency has been associated with idiopathic infantile arterialcalcification (IIAC), insulin resistance, hypophosphatemic rickets, andossification of the posterior longitudinal ligament of the spine.

IIAC, a rare autosomal recessive and nearly always fatal disorder, ischaracterized by calcification of the internal elastic lamina ofmuscular arteries and stenosis due to myointimal proliferation. Thereare more than 160 cases of IIAC that have been reported world-wide. Thesymptoms of the disease most often appear by early infancy, and thedisease is lethal by 6 months of age, generally because of ischemiccardiomyopathy, and other complications of obstructive arteriopathyincluding renal artery stenosis.

Although defects in the NPP1 protein have been implicated in suchserious disease as IIAC, currently no treatment is available for thosewho are affected by the disease and other calcification diseases causedby high total body burden of calcium and phosphorus due to abnormal bonemetabolism; low levels of circulating and locally produced inhibitors ofphosphate producers; or impaired renal excretion.

Current therapeutic options to prevent vascular calcification havelimited efficacy and undesirable and/or unacceptable side effects. Forexample, very large quantities of exogenous PPi are needed for efficacyand other inhibitors hydroxyapatite formation inhibit calcification ofbone and can lead to osteomalacia. In particular, direct administrationof exogenous PPi was found to prevent calcification in uremic animalmodels. (O'Neil, W. C. et al., Kidney Int 2011, 79, 512-517; Riser, B.L. et al., Nephrol Dial Transp 2011, 26, 3349-3357). But, this approachrequired high doses of PPi, due to the short half-life of PPi, andresulted in supraphysiologic plasma levels of PPi leading to localirritation. Bisphosphonates, which are non-hydrolyzable analogs of PPi,have been used to treat vascular calcification, e.g., in animal models.(Fleisch, H. et al., Europ J Clin Invest 1970, 1, 12-18; Price, P. A. etal., Arteriosclerosis Throm and Vas Bio 2001, 21, 817-824; Price, P. A.et al., Kidney Int 2006, 70, 1577-1583; Lomashvili, K. A. et al., KidneyInt 2009, 75, 617-625). However, bisphosphonates also inhibit boneformation. Bisphosphonates can delay but not stop calcification insubjects with GACI (Rutsch, F. et al., Circ Cardiovasc Genet 2008, 1,133-140), and, as in animals, lead to osteomalacia. (Otero, J. E., etal., J Bone Miner Res 2013, 28, 419-430).

Braddock, D. et al., (WO 2014/126965A2) discloses compositions andmethods for treating pathological calcification and ossification byadministering NPP1. Quinn, A. et al., (WO 2012/125182A1) discloses aNPP1 fusion protein to treat conditions including GACI, arterialcalcification, insulin resistance, hypophasphatemic rickets, andossificaiton of the posterior longitudinal ligament of the spine.

In spite of considerable research in the field, there is a continuingneed for new therapies to effectively inhibit vascular calicification,preferably without causing osteomalacia. There is also a need for aneffective and safe medicament for the treatment of IIAC, vascularcalcification in chronic kidney disease (VCCKD), pseudoxanthomaelasticum (PXE), insulin resistance, hypophosphatemic rickets, andossification of the posterior longitudinal ligament of the spine.

SUMMARY OF THE INVENTION

The present invention relates to uses of isolated recombinant humansoluble NPP1 that lacks N-terminal cytosolic and transmembrane domainsand fusion proteins thereof for the treatment of NPP1-deficiency orother progressive disorders characterized by the accumulation ofdeposits of calcium and other minerals.

The proteins of the invention can be surprisingly used to restore bloodNPP1 activity and restore normal level of pyrophosphate in subjectshaving deficiencies in NPP1 activity or exhibiting accumulation ofcalcium deposits in the bones, joints, heart, blood vessels, eyes,and/or the skin.

More specifically, the NPP1 proteins and NPP1 fusion proteins of theinvention can be used to treat subjects having NPP1-deficiency or otherdiseases or disorders associated with low levels of pyrophosphate,including but not limited to, idiopathic infantile arterialcalcification (IIAC, also known as general arterial calcification ininfants), vascular calcification in chronic kidney disease (VCCKD),pseudoxanthoma elasticum (PXE), insulin resistance, hypophosphatemicrickets, joint calcification, myocardial ischemia, and ossification ofthe posterior longitudinal ligament of the spine. Any progressivedisorder that is characterized by the accumulation of deposits ofcalcium and other minerals in arterial and/or connective tissues arewithin the scope of the present invention.

In some aspects, the invention relates to a method of reducing tissuecalcification, preferably vascular calcification in a subject in needthereof. The method comprises administering to a subject with low plasmapyrophosphate (PPi) or elevated inorganic phosphate (Pi), two or moredoses of a therapeutically effective amount of a composition comprisinga soluble ectonucleotide pyrophosphatase phosphodiesterase (NPP1). Eachdose contains an amount of soluble NPP1 that is sufficient to achieve atransient increase in plasma PPi in the subject. The transient increasein plasma PPi characterized by a peak PPi level that is at least about40% of the normal plasma PPi level and a return to base-line PPi levelwithin about 48 hours after administration of the dose. The time periodbetween doses is at least 2 days.

The transient increase in plasma PPi is maintained for at least about 4hours, preferably, at least about 6 hours, at least about 8 hours, atleast about 10 hours or at least about 12 hours.

The tissue calcification can be vascular calcification, such as venousor arterial calcification, and the calcification can be intimal ormedial.

The subject in need of therapy may have NPP1 deficiency, chronic kidneydisease (CKD), end-stage renal disease (ESRD), generalized arterialcalcification of infancy (GACI), cardiovascular disorder, diabetesmellitus II, atherosclerosis or pseudoxanthoma elasticum (PXE). When thesubject has low plasma PPi, the pretreatment levels of plasmapyrophosphate (PPi) in the subject is at least about 40% lower than thatof the normal plasma PPi levels and the subject is human. When thesubject has high levels of Pi, the pretreatment levels of Pi in thesubject are typically at least about 110% of the normal plasma Pilevels.

The amount of sNPP1 administered in each dose can be about 1.0 mg/kg toabout 5.0 mg/kg NPP1 or about 1.0 mg/kg to about 10.0 mg/kg NPP1. Thetime period between doses of NPP1 is at least 2 days and can be longer,for example at least 3 days, at least 1 week, 2 weeks or 1 month. ThesNPP1 can be administered in any suitable way, such as intravenously,subcutaneously, or intraperitoneally.

In preferred aspects, a NPP1 fusion protein is administered. Preferredfusion proteins comprise and NPP1 component an Fc region of animmunoglobulin and optionally a targeting moiety. A preferred targetingmoiety is Asp₁₀. Particularly preferred NPP1 fusion proteins foradministration in accordance with the methods disclosed herein have theamino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11 or SEQ ID NO:12.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the amino acid sequence of wild-type NPP1 protein (SEQ IDNO:1). The cytosolic and transmembrane regions are underlined. Thepotential N-glycosylation sites are in bold. The amino acid motif“PSCAKE” (SEQ ID NO:17) in bold is the start of a soluble NPP1 whichincludes the cysteine rich region.

FIG. 2 is the amino acid sequence of a sNPP1 that contains thecysteine-rich region, catalytic region and c-terminal region (SEQ IDNO:2).

FIG. 3 is the amino acid sequence of sNPP1-Fc fusion protein (SEQ IDNO:3).

FIG. 4 is the amino acid sequence of sNPP1-Fc-D10 (SEQ ID NO:4). The Fcsequence is underlined. The D10 (SEQ ID NO:18) targeting moiety is inbold.

FIG. 5 illustrates pyrophosphate levels in blood in wild-type mice afteradministration of sNPP1-Fc or sNPP1-FcD10 intravenously (1 hour postinjection) and subcutaneously (4 hour post injection).

FIG. 6 illustrates prevention of aortic calcification in Enpp1(−/−) micewith sNPP1-Fc-D10 treatment. Enpp1 (−/−) mice were treatedsubcutaneously with vehicle or 6 mg/kg sNPP1-Fc-D10 every other day overa period of 21 days. Aortic calcium levels are shown for males andfemales.

FIG. 7 illustrates blood PPi and enzymatic activity levels in Enpp1(−/−)mice treated with 6 mg/kg sNPP1-Fc-D10 intravenously. Plasma at timepoints of 0, 4, 24, 48, and 72 hours were collected and analyzed forNPP1 activity (dashed) and PPi levels (solid). The wild-type PPi levelwas determined to be 2.18 μM (data not shown). The dashed lines from topto bottom show the PPi levels for wild-type, heterozygous Enpp1(+/−),and homozygous Enpp1(−/=) asj mice (Li et. al, 2013). The profiles forsNPP1-Fc were similar to those of sNPP1-Fc-D10.

FIG. 8 illustrates increased survival of Enpp1^(asj) homozygous malemice treated with 5 mg/kg sNPP1-Fc in comparison to vehicle treatedmice. Wild-type and Enpp1^(asj) mice were placed on a high phosphorus,low magnesium diet starting at birth. Vehicle or sNPP1-Fc (5 mg/kg) wasdose subcutaneously every other day starting at 14 days of age.Kaplan-Meier survival curves showed that >50% of asj mice died prior to6 weeks, and all animals died by 9 weeks. In comparison, 50% of sNPP1-Fctreated animals survived past 7 week and are still living at 9 weeks.

FIGS. 9A and 9B illustrate increased percent body weight gain ofEnpp1^(asj) male mice treated with 5 mg/kg sNPP1-Fc (FIG. 9B) incomparison to vehicle treated mice (FIG. 9A). Wild-type and Enpp1^(asj)mice were placed on a high phosphorus, low magnesium diet starting atbirth. Vehicle or sNPP1-Fc (5 mg/kg) was injected subcutaneously everyother day starting at 14 days of age. Percent body weight gain forwild-type (solid line) and Enpp1^(asj) (circles) mice were plotted fromtwo to nine weeks of age. All Enpp1^(asj) animals were dead (opencircle) in the vehicle group at nine weeks (upper panel). In comparison,five Enpp1^(asj) mice were alive (solid circle) and five were dead (opencircle) in the sNPP1-Fc treatment group at the end of nine weeks.

FIGS. 10A-10C are photographs of wild-type (FIG. 10A, top), vehicletreated Enpp1^(asj) (FIG. 10B, middle) sNPP1-Fc treated (5 mg/Kg)treated Enpp1^(asj) (FIG. 10C, bottom) mice.

FIG. 11 illustrates levels of fibroblast growth factor 23 in vehicletreated wild-type, vehicle treated Enpp1^(asj/asj), and sNPP1-Fc treated(5 mg/Kg) Enpp1^(asj/asj) mice.

FIGS. 12A-12H are the amino acid sequences of soluble NPP1 compounds,fusion partners and fusion proteins. FIG. 12A shows the amino acidsequences of a soluble NPP1 containing amino acids from 107 to 925 ofSEQ ID NO:1 (SEQ ID NO:5). FIG. 12B shows the amino acid sequence of asoluble NPP1 containing amino acids from 187 to 925 of SEQ ID NO:1 (SEQID NO:6). FIG. 12C shows the amino acid sequence of the Fc region ofhuman IgG1 including the hinge region (SEQ ID NO:7). FIG. 12D shows theamino acid sequence of the Fc of human IgG1 including a partial hingeregion (SEQ ID NO:8). FIG. 12E shows the amino acid sequence of aNPP1-Fc fusion protein (SEQ ID NO:9). The NPP1 component contains SEQ IDNO:5, and the Fc sequence includes the hinge region. FIG. 12F shows theamino acid sequence of a NPP1-Fc fusion protein (SEQ ID NO:10). Thesoluble NPP1 contains SEQ ID NO:5, and the Fc sequence includes thepartial hinge region. FIG. 12G shows the amino acid sequence of aNPP1-Fc fusion protein (SEQ ID NO:11). The soluble NPP1 contains SEQ IDNO:6, and the Fc sequence includes the hinge region. FIG. 12H shows theamino acid sequence of a NPP1-Fc fusion protein (SEQ ID NO:12). Thesoluble NPP1 contains SEQ ID NO:6, and the Fc sequence includes thepartial hinge region.

FIGS. 13A-13C are autoradiogram of thin-layer chromatograms whichillustrates the activity of recombinant NPP1 in vitro and in vivo. FIG.13A: 100 nM ATP incubated with 130 ug/ml sNPP1-Fc-D10 for one hour at37° C. FIG. 13B: 100 nM ATP incubated with plasma from wild-type mice(WT), Enpp1^(−/−) mice, and Enpp1^(−/−) mice 2 hours after IV injectionof recombinant NPP1 (6 mg/kg). FIG. 13C: 100 nM ATP incubated with aortafrom wild-type mice (WT), Enpp1^(−/−) mice, and Enpp1^(−/−) mice 2 hoursafter IV injection of recombinant NPP1 (6 mg/kg). Pi: orthophosphate;ATP: Adenosine triphosphate; PPi: pyrophosphate.

FIGS. 14A and 14B are histograms which illustrates the time course ofplasma NPP1 activity (FIG. 14A, top) and plasma pyrophosphateconcentration (FIG. 14B, bottom) in Enpp1(−/−) mice after subcutaneousinjection of recombinant NPP1 (5 mg/kg).

FIG. 15 is a scatter-plot which illustrates the relationship betweenplasma NPP1 activity and plasma pyrophosphate (PPi) for Enpp1(−/−) miceat various times after subcutaneous injection of recombinant NPP1 (5mg/kg) (circles) and for wild-type mice (squares).

FIGS. 16A-16C are histograms which illustrates the synthesis ofpyrophosphate in human blood. FIG. 16A: Heparinized blood or plasmaobtained from the same blood sample. FIG. 16B: Centrifuged blood cellswith (all cells) or without buffy coat (erythrocytes) removed, suspendedin HEPES-buffered saline. FIG. 16C: Isolated leukocytes or platelets,suspended in HEPES-buffered saline. Samples were incubated at 37° C. for2 hours with or without recombinant NPP1 (145 ug/ml).

FIG. 17 is a histogram which illustrates the effect of recombinant NPP1on aortic calcification in Enpp1(−/−) mice. Recombinant NPP1 wasinjected (6 mg/kg) subcutaneously every 48 hours in mice fed with a highphosphate diet. Each bar represents a single animal with age in weeksgiven underneath. M: male pair; F: female pair. Dashed line indicatesthe mean calcium content of aortas from wild-type littermates.

FIG. 18 is a histogram which illustrates the effect of recombinant NPP1on aortic calcification in uremic rats with renal failure. sNPP1-Fc-D10or control was injected (5 mg/kg) subcutaneously, 5 dose per week for 21days in uremic rats fed with a high adenine diet. Each bar represents asingle animal aged approximately 4 months.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inpractice or testing of the present invention, the preferred methods andmaterials are described.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “altered PPi:Pi ratio” refers to a ratio of PPi in plasma to Piin serum that is at least 10% or at least 20% higher or lower than anormal PPi:Pi ratio for that type of subject (e.g. a human). An alteredPPi:Pi ratio can be present because of lower than normal levels ofplasma PPi or higher than normal levels of serum Pi. The ratio of PPi:Piis expressed as ([PPi]/[Pi])*1000, and the normal ratio of a human isabout 1.75.

As used herein, the term “fragment”, with regard to NPP1 proteins,refers to a subsequence of the full-length NPP1. A “fragment” of aprotein or peptide can be at least about 20 amino acids in length; forexample, at least about 50 amino acids in length; at least about 100amino acids in length; at least about 200 amino acids in length; atleast about 300 amino acids in length; or at least about 400 amino acidsin length (and any integer value in between). The fragments may range insize from four amino acid residues to the entire amino acid sequenceminus one amino acid. Thus, a protein “comprising at least a portion ofthe amino acid sequence of SEQ ID NO: 1” encompasses the full-lengthNPP1 and fragments thereof.

The term “high serum Pi” as used herein refers to a level of inorganicphosphate (Pi) in the serum of a subject that is at least 110% of thenormal level of Pi for that type of subject (e.g. a human). Preferably,the level of Pi in the serum of the subject at least about 120%, atleast about 150%, at least about 200% or at least about 300% of thenormal level of Pi for that type of subject. Normal Pi levels for ahuman are reported to be 1.5±0.5 millimolar (Rutsch, F. et al., CircCardiovasc Genet 1:133-140 (2008)).

An “isolated” or “purified” soluble NPP1 protein or biologically activefragment or fusion protein thereof is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue sourcefrom which the NPP1 protein, biologically active fragment or NPP1 fusionprotein is derived, or substantially free from chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of NPP1 protein,biologically active fragment, or NPP1 fusion protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. In one embodiment, the language“substantially free of cellular material” includes preparations of NPP1protein, biologically active fragment or NPP1 fusion protein having lessthan about 30% (by dry weight) of non-NPP1 protein/fragment/fusionprotein (also referred to herein as a “contaminating protein”), morepreferably less than about 20% of non-NPP1 protein/fragment/fusionprotein, still more preferably less than about 10% of non-NPP1protein/fragment/fusion protein, and most preferably less than about 5%non-NPP1 protein/fragment/fusion protein. When the NPP1 protein, fusionprotein, or biologically active fragment thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume of the protein preparation.

The term “low plasma PPi” as used herein refers to a level ofpyrophosphate (PPi) in the plasma of a subject that is no more than 50%of the normal level of PPi for that type of subject (e.g. a human).Preferably, the level of PPi in the plasma of the subject no more thanabout 40%, about 30%, about 20% or about 10% of the normal level of PPifor that type of subject. Normal PPi levels for a human are reported tobe 2.63±0.47 microMolar. (O'Neill et al., Nephrol Dial Transplant 2010,25, 187-191). Pyrophosphate can be quantified enzymatically usingsuitable known methods, such as the uridine-diphosphoglucose (UDPG)method. (Ryan, L. M. et al., Arthritis Rheum 1979, 22, 886-91).

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

As used herein, the term “subject” encompasses mammals and non-mammals.Examples of mammals include, but are not limited to, humans,chimpanzees, apes monkeys, cattle, horses, sheep, goats, swine, rabbits,dogs, cats, rats, mice, guinea pigs, and the like. Examples ofnon-mammals include, but are not limited to, birds, fish and the like.

As used herein, the term “therapeutically effective amount” refers to anontoxic but sufficient amount of an agent (e.g. sNPP1 proteins) which,as compared to a corresponding subject who has not received such amount,results in improved treatment, healing, prevention, or amelioration of adisease, disorder, or side effect, or a decrease in the rate ofadvancement of a disease or disorder. The term also includes within itsscope amounts effective to enhance normal physiological function. Theterm “treating” includes the application or administration of the NPP1proteins, fragments and fusion proteins of the invention to a subject,or application or administration of NPP1 proteins, fragments and fusionproteins of the invention to a subject who has an NPP1-associateddisease or disorder or other disease or disorder associated with lowlevels of blood pyrophosphate, or other progressive disorder that ischaracterized by the accumulation of deposits of calcium and otherminerals (mineralization), with the purpose of curing, healing,alleviating, relieving, altering, remedying, ameliorating, preventing,improving, or affecting the disease or disorder. The term “treating”refers to any indicia of success in the treatment or amelioration of aninjury, pathology or condition, including any objective or subjectiveparameter such as abatement; remission; diminishing of symptoms ormaking the injury, pathology or condition more tolerable to the subject;slowing in the rate of degeneration or decline; making the final pointof degeneration less debilitating; or improving a subject's physical ormental well-being. Treatment may be therapeutic or prophylactic. Thetreatment or amelioration of symptoms can be based on objective orsubjective parameters; including the results of a physical examination.

Methods of Treatment

The present invention relates to uses of an isolated recombinant humansoluble NPP1 (“sNPP1”) which lacks an N-terminal portion (i.e., lackingcytosolic and transmembrane domains) and fusion proteins thereof for thetreatment of NPP1-associated diseases and disorders. The proteins of theinvention can be surprisingly used to increase NPP1 activity in vivo andincrease or restore normal level of blood pyrophosphate (PPi) insubjects. The proteins of the invention can be also used to preventaccumulation of deposits of calcium in joints, kidney, heart (e.g.,aorta), artery, blood vessels, or posterior longitudinal ligament of thespine.

The subject can be a human patient having deficiencies in NPP1 activity(NPP1 deficiency) exhibiting low levels of pyrophosphate, suffering froma disease or disorder associated with low levels of pyrophosphate, orsuffering from a progressive disorder that is characterized by theaccumulation of deposits of calcium and other minerals (mineralization)in elastic fibers. Mineralization can occur at the heart, arteries,blood vessels, the kidney, the ligaments of spine, the skin, eyes, andthe digestive tract.

More specifically, the NPP1 proteins and NPP1 fusion proteins of theinvention can be used to treat subjects having NPP1-associated diseasesor disorders, including but not limited to, idiopathic infantilearterial calcification (IIAC), insulin resistance, hypophosphatemicrickets, and ossification of the posterior longitudinal ligament of thespine, or other diseases such as vascular calcification in chronickidney disease (VCCKD), myocardial ischemia, joint calcification,angioid streaks, and pseudoxanthoma elasticum (PXE).

The soluble NPP1 proteins, fragment, and NPP1 fusion proteins thereofcan be used to treat a wide variety of conditions in a subject. Forexample, treatment of conditions that can be improved by reducing and/oreliminating one or more calcification structures and/or preventingcalcification structures from forming in a subject such as a mammal, forexample, a human patient is within the scope of the invention.

In one particularly useful embodiment, the condition to be treated isgeneralized arterial calcification (also known as idiopathic arterialcalcification of infancy and arterial media calcification of infancy).

In other embodiments, conditions such as pseudoxanthoma elasticum,vascular calcification in chronic kidney disease, insulin resistance,hypophosphatemic rickets, or ossification of the posterior longitudinalligament of the spine can be also treated using the methods describedherein.

Generally, the dosage of fusion protein administered to a subject willvary depending upon known factors such as age, health and weight of therecipient, type of concurrent treatment, frequency of treatment, and thelike. Usually, a dosage of active ingredient (i.e., fusion protein) canbe between about 0.0001 and about 50 milligrams per kilogram of bodyweight. Precise dosage, frequency of administration and time span oftreatment can be determined by a physician skilled in the art ofadministration of therapeutic proteins.

A preferred embodiment of the present invention involves a method fortreatment of an NPP1-associated disease or other calcification diseaseswhich includes the step of administering a therapeutically effectiveamount of an isolated soluble NPP1 protein (sNPP1), biologically activefragment, or NPP1 fusion protein to a subject. As defined herein, atherapeutically effective amount of protein (i.e., an effective dosage)ranges from about 0.001 to 50 mg/kg body weight. The skilled artisanwill appreciate that certain factors may influence the dosage requiredto effectively treat a subject, including but not limited to theseverity of the disease, previous treatments, the general health and/orage of the subject, and other diseases present. Moreover, treatment of asubject with a therapeutically effective amount of protein can include asingle treatment or, preferably, can include a series of treatments. Itwill also be appreciated that the effective dosage of protein used fortreatment may increase or decrease over the course of a particulartreatment.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 50mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a protein, polypeptide, orantibody can include a single treatment or, preferably, can include aseries of treatments.

In a preferred example, in the range of between about 0.1 to 20 mg/kgbody weight, one time per week, twice per week, once in about 10 days,once in about 12 days, once in about 14 days, once in about 17 days,once in about 20 days, once in about 25 days, or once in about 30 days.It will also be appreciated that the effective dosage of soluble sNPP1protein, biologically active fragment or fusion protein thereof used forthe treatment may increase or decrease over the course of a particulartreatment.

The invention provides for a therapeutically effective dose of sNPP1,biologically active fragment or fusion protein thereof to beadministered to a patient between one time every 5 days and one timeevery 30 days for a period of time determined by a practitioner of skillin the art of medical sciences. In one embodiment, the period of timewill be the remainder of the patient's life span. In one embodiment, thedosing frequency is between one time every 5 days and one time every 25days. In one embodiment, the dosing frequency is between one time every5 days and one time every 21 days. In another embodiment, the dosingfrequency is between one time every 7 days and one time every 14 days.sNPP1, biologically active fragment or fusion protein thereof can beadministered one time every 5 days, one time every 6 days, one timeevery 7 days, one time every 8 days, one time every 9 days, one timeevery 10 days, one time every 11 days, one time every 12 days, one timeevery 13 days, or one time every 14 days. In some embodiments, sNPP1,biologically active fragment or fusion protein thereof is administeredabout weekly. In other embodiments, sNPP1, biologically active fragmentor fusion protein thereof is administered about bi-weekly. In oneembodiment, the dosing frequency is one time about 30 days.

In one embodiment, the patient is less than 2 years of age. In someembodiments, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg,about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 5 mg, about 10mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg,about 40 mg, or about 45 mg of sNPP1, biologically active fragment orfusion protein is administered to the patient with NPP1-deficiency orother calcification disease. In some embodiments, about 0.5 to about 30mg, about 0.5 to about 20 mg, about 0.5 to about 10 mg, or about 0.5 toabout 5 mg are administered to the patient.

In one embodiment, about 1 mg/kg of sNPP1, biologically active fragmentor fusion protein is administered to the patient once a week. In oneembodiment, about 2 mg/kg of sNPP1, biologically active fragment orfusion protein is administered to the patient once a week. In oneembodiment, about 3 mg/kg of sNPP1, biologically active fragment orfusion protein is administered to the patient once a week. In oneembodiment, about 4 mg/kg of sNPP1, biologically active fragment orfusion protein is administered to the patient once a week. In oneembodiment, about 5 mg/kg of sNPP1, biologically active fragment orfusion protein is administered to the patient once a week. In oneembodiment, about 6 mg/kg of sNPP1, biologically active fragment orfusion protein is administered to the patient once a week. In oneembodiment, about 7 mg/kg of sNPP1, biologically active fragment orfusion protein is administered to the patient once a week. In oneembodiment, about 8 mg/kg of sNPP1, biologically active fragment orfusion protein is administered to the patient once a week. In oneembodiment, about 9 mg/kg of sNPP1, biologically active fragment orfusion protein is administered to the patient once a week. In oneembodiment, about 10 mg/kg of sNPP1, biologically active fragment orfusion protein is administered to the patient once a week.

In some embodiments, the level of blood PPi in a patient prior totreatment is about 1%, about 2%, about 3%, about 5%, about 10%, about15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,or about 80% of normal levels of PPi observed in a normal humanindividual. In one embodiment, the level of PPi in a patient prior totreatment is about 50% or less of normal levels of PPi observed in anormal human individual. In one embodiment, the level of PPi in apatient prior to treatment is about 40% or less of normal levels of PPiobserved in a normal human individual. In some embodiments, the level ofPPi in a patient prior to treatment is about 30% or less of normallevels of PPi observed in a normal human individual. In someembodiments, the level of PPi in a patient prior to treatment is about30% or less of normal levels PPi observed in a normal human individual.In some embodiments, the level of PPi in a patient prior to treatment isabout 20% or less of normal levels of PPi observed in a normal humanindividual. In some embodiments, the level of PPi in a patient prior totreatment is about 10% or less of normal levels of PPi observed in anormal human individual. In some embodiments, the level of PPi in apatient prior to treatment is about 5% or less of normal levels of PPiobserved in a normal human individual. In some embodiments, a patientshows no measurable PPi prior to treatment.

sNPP1, biologically active fragment or fusion protein can beadministered by, for example, subcutaneous injections, intramuscularinjections, and intravenous (IV) infusions or injections.

In one embodiment, sNPP1, biologically active fragment or fusion proteinis administered intravenously by IV infusion by any useful method. Inone example, sNPP1, biologically active fragment or fusion protein canbe administered by intravenous infusion through a peripheral line. Inanother example, sNPP1, biologically active fragment or fusion proteincan be administered by intravenous infusion through a peripherallyinserted central catheter.

In another embodiment, sNPP1, biologically active fragment or fusionprotein is administered intravenously by IV injection.

In another embodiment, sNPP1, biologically active fragment or fusionprotein can be administered via intraperitoneal injection.

In another embodiment, sNPP1, biologically active fragment or fusionprotein can be administered by subcutaneous injections.

In another embodiment, sNPP1, biologically active fragment or fusionprotein can be administered by intramuscular injections.

In still another embodiment, sNPP1, biologically active fragment orfusion protein is administered via a pharmaceutically acceptable capsuleof the therapeutic protein. For example, the capsule can be anenteric-coated gelatin capsule.

In one embodiment, the method involves administering the soluble NPP1protein or NPP1 fusion protein of the invention alone, or in combinationwith other agent(s). In one embodiment, the method involvesadministering an NPP1 protein or an NPP1 fusion protein of the inventionas therapy to compensate for reduced or aberrant NPP1 expression oractivity in the subject having an NPP1-deficiency or other associateddisease or disorder.

In one embodiment, the isolated sNPP1 proteins, fragments, and fusionproteins can be administered before, after or concurrently with theagent or can be co-administered with other known therapies.Co-administration of the isolated sNPP1 proteins, fragments, and fusionproteins of the present invention with other therapeutic agents mayprovide two agents which operate via different mechanisms which yield anincreased therapeutic effect. Such co-administration can solve problemsdue to development of resistance to drugs.

In particular aspects, this disclosure relates to a method for reducingvascular calcification in a subject in need thereof. The method is basedon the surprising finding that soluble forms of NPP1 can be administeredto animals that have low plasma PPi levels (an inhibitor or tissuecalcification) or high serum Pi levels, to cause a transient increase inplasma PPi in the animals, and that the transient increase in plasma PPican inhibit vascular calcification in the animal. Since the increase inplasma PPi is transient, therapy can be tailored to inhibit undesirableor pathological tissue calcification, such as vascular calcification,without inhibiting bone calcification or inducing osteomalacia.

In general terms, the disclosure relates to a method for reducing tissuecalcification (e.g., vascular calcification) in a subject in needthereof, by administering to the subject two or more doses of solubleNPP1 (sNPP1). Each of the doses contains an amount of soluble NPP1 thatis sufficient to achieve a transient increase in plasma PPi in thesubject, preferably with a return to base-line PPi level within about 48hours after administration of the dose. The time period between theadministration of each dose is generally at least 2 days.

The subject in need thereof can be of any age and gender, and preferablyhas low plasma PPi or high serum Pi (e.g., resulting in an alteredPPi:Pi ratio). Low plasma PPi can be due, for example, to congenitalNPP1 deficiency such as mutation in the gene encoding NPP1 that lead toreduced expression of active NPP1 or reduced enzymatic activity(associated with NPP1 deficiency and autosomal-recessivehypophosphatemic rickets), and mutation in the gene encoding MRP6 thatlead to absent or nonfunctional MRP6 protein (associated withpseudoxanthoma elasticum). Low plasma PPi or high serum Pi is alsofrequently seen in patients with chronic kidney disease, end-stage renaldisease/failure, diabetes mellitus and other conditions. Accordingly,the subject in need of therapy can have chronic kidney disease (CKD),end-stage renal disease (ESRD), generalized arterial calcification ofinfancy (GACI), diabetes mellitus II, autosomal-recessivehypophosphatemic rickets, a cardiovascular disorder, atherosclerosisand/or pseudoxanthoma elasticum (PXE). The subject is generally a human,but can also be any other suitable mammal or non-mammal.

Tissue calcification is a progressive process and individuals born withcongenital NPP1 deficiency may not show calcification of tissues forseveral years. By initiating therapy as early as possible, it is likelythat calcification can be reduced and or minimized in such subjects. Insubjects with low plasma PPi levels not caused by germ line mutation, orwith high serum Pi levels (e.g., with an altered plasma PPi:Pi ratio),therapy should begin as soon as practicable (i.e., soon after thediagnosis of the conditions, such as chronic kidney disease (CKD) orend-stage renal disease (ESRD)). In certain embodiments, the subject tobe treated can be between 1 month and 24 months in age, less than 1 yearof age, less than 2 years of age, less than 3 years of age, less than 4years of age, or less than 5 years of age.

Each dose of sNPP1 that is administered to the subject contains anamount of sNPP1 sufficient to achieve a transient increase in plasmaPPi. Preferably, the transient increase is characterized by a peak PPilevel that is at least about 40% of the normal plasma PPi level, atleast about 50% of the normal plasma PPi level, at least about 60% ofthe normal plasma PPi level, at least about 70% of the normal plasma PPilevel, at least about 80% of the normal plasma PPi level, between about40% and 100% of the normal plasma PPi level, between about 50% and 100%of the normal plasma PPi level, between about 60% and 100% of the normalplasma PPi level, between about 70% and 100% of the normal plasma PPilevel, between about 80% and 100% of the normal plasma PPi level, orbetween about 100% and 200% of the normal plasma PPi level.

Preferably, the transient increase in plasma PPi after administration ofsNPP1 is maintained for at least about 4 hours, at least about 6 hours,at least about 8 hours, at least about 10 hours or at least about 12hours. In addition, it is preferred that the transient increase inplasma PPi returns to the subject's base-line PPi level within about 48hours after administration of the dose, within about 3 days afteradministration of the dose or within about 4 days after administrationof the dose.

The low plasma PPi in a subject prior to treatment is about 50% or less,preferably 40% or less of normal levels of PPi observed in a normalsubject (e.g., a human). In some aspects, the level of PPi in a subjectprior to treatment is about 30% or less of normal levels of PPi. Inother aspects, the level of PPi in a subject prior to treatment is about20% or less of normal levels of PPi. In some other aspects, the level ofPPi in a subject prior to treatment is about 10% or less of normallevels. In some aspects, a subject may have no measurable PPi prior totreatment.

The high serum Pi in a subject prior to treatment is about 110% or more,preferably 125% or more of normal levels of Pi observed in a normalsubject (e.g., a human). In some aspects, the level of Pi in a subjectprior to treatment is about 150% or more of normal levels of PPi. Inother aspects, the level of Pi in a subject prior to treatment is about200% or more of normal levels of PPi. In some other aspects, the levelof Pi in a subject prior to treatment is about 300% or more of normallevels. Without wishing to be bound by any particular theory, it isbelieved that inducing a transient increase in serum PPi can compensatefor elevated plasma Pi levels and transiently restore normal or nearlynormal PPi:Pi ratio, thereby inhibiting tissue calcification which ispromoted by higher than normal levels of serum Pi.

The amount of sNPP1 sufficient to achieve the transient increase inplasma PPi can be easily determined by a clinician of ordinary skill,for example, by administering a dose that is expected to produce thetransient increase in plasma PPi, determining whether the transientincrease occurs and then making appropriate adjustments to the dose. Theamount to administer will be influenced by a number of conventionalfactors, including the particular sNPP1 used, the age, health and weightof the subject, the subject's sensitivity to drugs, and other relevantfactors. Typically, the amount of sNPP1 to be administered in each doseis between about 0.001 and about 50 milligrams per kilogram of bodyweight, with 1 mg/kg to 5 mg/kg, 1 mg/kg to 10 mg/kg, 1 mg/kg to 20mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, or 20 mg/kg beingpreferred. Precise dosage, frequency of administration and time span oftreatment can be determined by a physician skilled in the art ofadministration of therapeutic proteins.

In some preferred embodiments, each dose contains about 1.0 mg to about5.0 mg sNPP1 per Kg body weight, about 1.0 mg to about 10.0 mg sNPP1 perKg body weight or about 1.0 mg to about 20.0 mg sNPP1 per Kg bodyweight.

The time period between doses is selected to permit the subject's serumPPi levels to return to base-line levels, and is at least 2 (48 hours)days, but can be longer as desired or indicated. For example, the timeperiod between doses can be 3 days, 4 days, 5 days, 6 days, one week, 10days, 12 days, two weeks, three weeks or about 1 month.

In general, it is desirable to initiate the therapy according to themethods described herein as soon as practicable after diagnosis of lowplasma PPi, high serum Pi, or NPP1 deficiency. Subjects born withcongenital NPP1 deficiency may not show calcification of tissues forseveral years. By initiating therapy as early as possible, it is likelythat calcification can be reduced and or minimized in such subjects. Insubjects with low plasma PPi levels not caused by germ line mutation orwith high serum Pi, therapy should begin as soon as practicable afterthe diagnosis of conditions, such as chronic kidney disease (CKD) orend-stage renal disease (ESRD).

The method provides an effective way to reduce tissue calcification(e.g. vascular calcification) in a subject with low plasma PPi or withhigh serum Pi, including those with an altered ratio of PPi to Pi. Thetissue calcification is preferably vascular calcification, which ispreferably arterial calcification but can also be venus calcification.The vascular calcification can be intimal or medial. The subject to betreated in accordance with the methods described herein can have NPP1deficiency, generalized arterial calcification (GACI), also known asidiopathic arterial calcification of infancy and arterial mediacalcification of infancy. The subject to be treated can also have acardiovascular disorder, such as coronary artery disease and/oratherosclerosis. The subject to be treated can have chronic kidneydisease (CKD) or end-stage renal disease (ESRD). The subject to betreated can have diabetes mellitus (e.g. type II diabetes). The subjectto be treated can have pseudoxanthoma elasticum (PXE).

The sNPP1 can be administered by any suitable method or route ofadministration, such as parenterally, orally or by inhalation.Parenteral administration, such as, intravenous injection or infusion,subcutaneous injection, intraperitoneal injections, or intramuscularinjections is preferred.

If desired, the sNPP1 can be administered with one or moreco-therapeutic agents. For co-therapy the sNPP1 and one or moreadditional therapeutic agents are administered so that there issubstantial overlap in their individual pharmacological activities inthe subject. Accordingly, any co-therapeutic agent can be administeredprior to, concurrently with or subsequent to the administration ofsNPP1. Co-therapy may provide two agents which operate via differentmechanisms which yield an increased therapeutic effect.

In addition to causing a transient increase in serum PPi, it is believedthat administering sNPP1 in accordance with the methods describedherein, can alter the levels of certain proteins in the subject. Forexample, without wishing to be bound by any particular theory, it isbelieved that administering sNPP1 in accordance with the methodsdescribed herein can decrease the levels of osteopontin, osteoprotegerinand fibroblast growth factor 23 (FGF-23) in the subject. The levels ofthese proteins can therefor also be used, in addition to plasma PPi andserum Pi levels, to monitor therapy and tailor dosing.

sNPP1

The present invention employes soluble NPP1 that a biologically activeNPP1 domain of NPP1 (i.e., NPP1 components that contain at least oneextracellular catalytic domain of naturally occurring NPP1 for thepyrophosphatase and/or phosphodiesterase activity). The soluble NPP1proteins of the invention comprise at least the NPP1 domain essential tocarry out the pyrophosphatase and/or phosphodiesterase activity.

In one embodiment, the soluble NPP1, fragment, and fusion proteinsthereof can form functional homodimers or monomer. In a preferredembodiment, a soluble NPP1 protein or NPP1 fusion protein thereof can beassayed for pyrophosphatase activity as well as the ability to increasepyrophosphate levels in vivo.

Preferred soluble NPP1 proteins and NPP1 fusion proteins of theinvention are enzymatically active in vivo (e.g., human). In oneembodiment, the soluble protein comprises amino acid sequence having atleast 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%sequence identity to the following sequence:

(SEQ ID NO: 2) PSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEKRLTRSLCACSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYLHTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASFSLKSKEKFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGSVPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDVPDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTPAPNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQFNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRNDSFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWRYFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIRNQEILIPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELLMLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQED

Any desired enzymatically active form of soluble NPP1 can be used in themethods described herein. The enzymatically active sNPP1 can increasePPi levels in suitable enzymatic assays, and can be assayed forpyrophosphatase activity, phosphodiesterase activity, or pyrophosphataseand phosphodiesterase activity. Typically, the sNPP1 contains at leastan NPP1 component that lacks the N-terminal cytosolic and transmembranedomains of naturally occurring transmembrane NPP1. In preferred aspects,the NPP1 component contains the cysteine-rich region (amino acids 99-204of SEQ ID NO:1) and the catalytic region (amino acids 205-591 of SEQ IDNO:1) of naturally occurring human NPP1. Typically, the NPP1 componentalso includes the C-terminal region (amino acids 592 to 925 of SEQ IDNO:1), and has the amino acid sequence of SEQ ID NO:2. However, theC-terminal region can be truncated if desired. Accordingly, preferredNPP1 components include the cysteine-rich region and catalytic region ofhuman NPP1 (amino acids 99-591 of SEQ ID NO:1) or the cysteine-richregion, the catalytic region and the C-terminal region of human NPP1(SEQ ID NO:2). Other preferred NPP1 components contain only a portion ofthe cysteine-rich domain and have the sequence of amino acids 107 to 925of SEQ ID NO:1 or amino acids 187 to 925 of SEQ ID NO:1.

The cysteine rich region of NPP1 (i.e., amino acids 99 to 204 of SEQ IDNO: 1) can facilitate dimerization of the sNPP1. The sNPP1, includingfusion proteins, can be in the form of a monomer of functionalhomodimer.

The amino acid sequence of the NPP1 component can be a variant of thenaturally occurring NPP1 sequence, provided that the NPP1 component isenzymatically active. NPP1 variants are enzymatically active and have atleast 80%, at least 85%, at least 90%, at least 95% and more preferablyat least 96% amino acid sequence identity to the corresponding portionof human NPP1 (e.g., over the length of the cysteine-rich region, thecatalytic region, the c-terminal region, the cysteine-rich region plusthe catalytic region, the cystein-rich region plus the catalytic regionplus the c-terminal region. Preferred NPP1 variants have at least 90%,preferably at least 95%, more preferably at least 97% amino acidsequence identity to (i) the amino acid sequence of residues 205-591 ofSEQ ID NO: 1, (ii) the amino acid sequence of residues 99-591 of SEQ IDNO:1, (iii) the amino acid sequence of residues 99-925 of SEQ ID NO:1,(iv) the amino acid sequence of residues 107-925 of SEQ ID NO:1, or (v)the amino acid sequence of residues 187-925 of SEQ ID NO:1. Suitablepositions for amino acid variation are well-known from NPP1 structuralstudies and analysis of disease-associated mutations in NPP1. Forexample, substitution of the following amino acids occurs in certaindisease-associated mutations that reduce NPP1 enzymatic activity, andvariations of the amino acids at these positions should be avoided:Ser216, Gly242, Pro250, Gly266, Pro305, Arg349, Tyr371, Arg456, Tyr471,His500, Ser504, Tyr513, Asp538, Tyr570, Lys579, Gly586; Tyr659, Glu668,Cys726, Arg774, His777, Asn792, Asp804, Arg821, Arg888, and Tyr901.(See, e.g., Jansen, S. et al., Structure 20:1948-1959 (2012).)

In one embodiment, the soluble NPP1 protein can be a fusion proteinrecombinantly fused or chemically bonded (e.g., covalent bond, ionicbond, hydrophobic bond and Van der Waals force) to a fusion partner. Inanother embodiment, the fusion protein has at least 70, 75, 80, 85, 90,91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:3or SEQ ID NO:4.

To determine the percent identity of two amino acid sequences, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). In a preferred embodiment, thelength of a reference sequence aligned for comparison purposes is atleast 30%, preferably at least 40%, more preferably at least 50%, evenmore preferably at least 60%, and even more preferably at least 70%,80%, or 90% of the length of the reference sequence (e.g., sNPP1 aminoacid sequence of SEQ ID NO:2; amino acids 107-925 of SEQ ID NO:1 oramino acids 187-925 of SEQ ID NO:1). The amino acid residues ornucleotides at corresponding amino acid positions are then compared.When a position in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid is equivalent to amino acid or nucleic acid“homology”). The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J MolBiol 1970, 48, 444-453) algorithm which has been incorporated into theGAP program in the GCG software package (available at www.gcg.com),using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.In another embodiment, the percent identity between two amino acid isdetermined using the algorithm of E. Meyers and W. Miller (CABIOS, 1989,4, 11-17) which has been incorporated into the ALIGN program (version2.0 or 2.0U), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The sNPP1 can consist of or consist essentially of an NPP1 component asdescribed herein. Alternatively, the sNPP1 can be in the form of afusion protein that contains an NPP1 component and one or more otherpolypeptides, referred to as fusion partners, optionally through asuitable linker in each instance, or in the form of a conjugate betweenan NPP1 component and another molecule (e.g., PEG). When the sNPP1 is inthe form of a fusion protein, each fusion partner is preferably locatedc-terminally to the NPP1 component. Without wishing to be bound by anyparticular theory, it is believed that fusion proteins that contain anNPP1 component that contains the cysteine-rich region and catalyticregion, and one or more fusion proteins that are located c-terminally tothe NPP1 component, are preferred over other configurations of NPP1fusion proteins because they can be expressed at sufficient levels andare sufficiently stable to be used as therapeutic proteins.

Any suitable fusion partner can be included in the fusion protein.Advantageously, a number of fusion partners are well-known in the artthat can provide certain advantages, such as reduced aggregation andimmunogenicity, increased the solubility, improved expression and/orstability, and improved pharmacokinetic and/or pharmacodynamicsperformance. See, e.g., Strohl, W. R. BioDrugs 29:215-239 (2015). Forexample, it is well-known that albumin, albumin fragments or albuminvariants (e.g., human serum albumin and fragments or variants thereof)can be incorporated into fusion proteins and that such fusion proteinscan be easily purified, stable and have an improved plasma half-life.Suitable albumin, albumin fragments and albumin variants that can beused in the sNPP1 fusion proteins are disclosed, for example in WO2005/077042A2 and WO 03/076567A2, each of which is incorporated hereinby reference in its entirety. Fusions to human transferrin are alsoknown to improve half-life. See, e.g., Kim B J et al., J Pharmacol ExprTher 334(3):682-692 (2010); and WO 2000/020746. Peptides that bind toalbumin or transferrin, such as antibodies or antibody fragments, canalso be used. See, e.g., EP 0486525 B1, U.S. Pat. No. 6,267,964 B1, WO04/001064A2, WO 02/076489A1, WO 01/45746, WO 2006/004603, and WO2008/028977. Similarly immunoglobulin Fc fusion proteins are well-known.See, e.g., Czajkowsky D M et al., EMBO Mol Med 4(10):1015-1028 (2012),U.S. Pat. Nos. 7,902,151; and 7,858,297, the entire teachings of whichare incorporated herein by reference in their entirety. The fusionprotein can also include a CTP sequence (see also, Fares et al.,Endocrinol 2010, 151, 4410-4417; Fares et al., Proc Natl Acad Sci 1992,89, 4304-4308; and Furuhashi et al., Mol Endocrinol 1995, 9, 54-63).Preferably, the fusion partner is the Fc of an immunoglobulin (e.g., Fcor human IgG1). The Fc can include CH1, CH2 and CH3 of human IgG1, andoptionally the human IgG1 hinge region (EPKSCDKTHTCPPCP (SEQ ID NO:13))or a portion of the human IgG1 hinge region (e.g., DKTHTCPPCP (SEQ IDNO:14) or PKSCDKTHTCPPCP (SEQ ID NO:15)) if desired. In some fusionproteins, the Fc can include CH2 and CH3 of human IgG1, or the Fc ofhuman IgG2 or human IgG4, if desired.

Preferably, the sNPP1 fusion protein comprises an NPP1 component and apeptide that increases the half-life of the fusion protein, mostpreferably the Fc of an immunoglobulin (e.g., Fc or human IgG1). As usedherein, a “protein that increases the half-life of the fusion protein”refers to a protein that, when fused to a soluble NPP1 or biologicallyactive fragment, increases the half-life of the soluble NPP1 polypeptideor biologically active fragment as compared to the half-life of thesoluble NPP1 polypeptide, alone, or the NPP1 biologically activefragment, alone.

In one embodiment, the half-life of the NPP1 fusion protein is increased50% as compared to the half-life of the NPP1 polypeptide or biologicallyactive fragment, alone. In another embodiment, the half-life of the NPP1fusion protein is increased 60% as compared to the half-life of the NPP1polypeptide or biologically active fragment, alone. In anotherembodiment, the half-life of the NPP1 fusion protein is increased 70% ascompared to the half-life of the NPP1 polypeptide or biologically activefragment, alone. In another embodiment, the half-life of the NPP1 fusionprotein is increased 80% as compared to the half-life of the NPP1polypeptide or biologically active fragment, alone. In anotherembodiment, the half-life of the NPP1 fusion protein is increased 90% ascompared to the half-life of the NPP1 polypeptide or biologically activefragment, alone.

In another embodiment, the half-life of the NPP1 fusion protein isincreased 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9fold, or 10 fold as compared to the half-life of the NPP1 polypeptide orbiologically active fragment, alone. Methods for determining thehalf-life of a protein or fusion protein are well known in the art. Forexample, Zhou et al., Determining Protein Half-Lives, Methods inMolecular Biology 2004, 284, 67-77 discloses numerous methods fortesting of the half-life of a protein. If desired, the fusion proteincan be conjugated to polymers or other suitable compounds that extendhalf-life, such as polyethylene glycol (PEG), can be conjugated to theNPP1 fusion proteins.

In one embodiment, the peptide which increases the half-life of thefusion protein is a CTP sequence (see also, Fares et al., 2010,Endocrinol., 151(9):4410-4417; Fares et al., 1992, Proc. Natl. Acad.Sci, 89(10):4304-4308; and Furuhashi et al., 1995, Molec. Endocrinol.,9(1):54-63).

In another embodiment, the peptide which increases the half-life of thefusion protein is an Fc domain of an Ig.

Fusion partners may also be selected to target the fusion protein todesired sites of clinical or biological importance (e.g., site ofcalcification). For example, peptides that have high affinity to thebone are described in U.S. Pat. No. 7,323,542, the entire teachings ofwhich are incorporated herein by reference. Peptides that can increaseprotein targeting to calcification sites can contain a consecutivestretch of at least about 4 acidic amino acids, for example, glutamicacids or aspartic acids. Typically, the peptide that targets the fusionprotein to calcification sites will comprise between 4 and 20consecutive acidic amino acids, for example 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive amino acids selectedfrom glutamic acid and aspartic acid. The peptide can consist solely ofglutamic acid residues, solely of aspartic acid residues, or be amixture of glutamic acid and aspartic acid residues. A particularlypreferred moiety for targeting to sights of calcification is Asp₁₀ (SEQID NO:18).

In one embodiment, the NPP1 fusion protein of the invention comprises anNPP1 polypeptide and a moiety that increase protein targeting tocalcification sites such as a consecutive stretch of acidic amino acids,for example, glutamic acids or aspartic acids.

Suitable peptide linkers for use in fusion proteins are well-known andtypically adopt a flexible extended conformation and do not interferewith the function of the NPP1 component or the fusion partners. Peptidelinker sequences may contain Gly, His, Asn and Ser residues in anycombination. The useful peptide linkers include, without limitation,poly-Gly, poly-His, poly-Asn, or poly-Ser. Other near neutral aminoacids, such as Thr and Ala can be also used in the linker sequence.Amino acid sequences which can be usefully employed as linkers includethose disclosed in Maratea et al., Gene 1985, 40, 39-46; Murphy et al.,Proc Natl Acad Sci USA 1986, 83, 8258-8262; U.S. Pat. Nos. 4,935,233 and4,751,180. Other suitable linkers can be obtained from naturallyoccurring proteins, such as the hinge region of an immunoglobulin. Apreferred synthetic linker is (Gly₄Ser)_(n), where n is 1, 2, 3, 4, 5,6, 7, 8, 9 or 10 (SEQ ID NO:19). Preferably, n is 3 or 4. For example,in some embodiments the linker is (Gly₄Ser)₃ (SEQ ID NO:16) and thefusion protein include a linker with the amino acid sequenceGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer (SEQ ID NO:16). Typically,the linker is from 1 to about 50 amino acid residues in length, or 1 toabout 25 amino acids in length. Frequently, the linker is between about8 and about 20 amino acids in length.

Preferred NPP1 fusion proteins comprise from N-terminus to C-terminus anNPP1 component, optionally a linker, an Fc region of an immunoglobulin(e.g., human IgG1 Fc optionally including hinge or a portion thereof),optionally a second liner, and optionally a targeting moiety. Thus, theFc region and the optional targeting moiety, when present, are eachlocated C-terminally to the NPP1 component. The NPP1 componentpreferably comprises the cysteine-rich region and the catalytic domainof NPP1, lacks the N-terminal cytosolic and transmembrane domains, andoptionally contains the C-terminal region.

A preferred fusion protein comprises, from N-terminus to C-terminus, anNPP1 component comprising the cysteine-rich domain, the catalytic domainand the C-terminal region of human NPP1; and the Fc region, includinghinge, of a human immunoglobulin. Preferably, the Fc region is fromhuman IgG1. In particular embodiments, the fusion protein has at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to SEQ ID NO:3. A preferredfusion protein of this type has the amino acid sequence of SEQ ID NO:3.

Another preferred fusion protein comprises, from N-terminus toC-terminus, an NPP1 component comprising the cysteine-rich domain, thecatalytic domain and the C-terminal region of human NPP1; a linker(e.g., (Gly₄Ser)₃ (SEQ ID NO:16)); and the Fc region, including hinge,of a human immunoglobulin. Preferably, the Fc region is from human IgG1.

Another preferred fusion protein comprises, from N-terminus toC-terminus, an NPP1 component comprising the cysteine-rich domain, thecatalytic domain and the c-terminal region of human NPP1; the Fc region,including hinge or a portion thereof, of a human immunoglobulin; and amoiety targeting the fusion protein to sites of calcification.Preferably, the Fc region is from human IgG1. Preferably, the moietytargeting the fusion protein to sites of calcification is Asp₁₀ (SEQ IDNO:18). More preferably, the Fc region is from human IgG1 and the moietytargeting the fusion protein to sites of calcification is Asp₁₀ (SEQ IDNO:18). In particular embodiments, the fusion protein has at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to SEQ ID NO:4. A preferred fusion proteinof this type has the amino acid sequence of SEQ ID NO:4.

Another preferred fusion protein comprises, from N-terminus toC-terminus, an NPP1 component comprising the cysteine-rich domain, thecatalytic domain and the c-terminal region of human NPP1; a linker(e.g., (Gly₄Ser)₃ (SEQ ID NO:16)); the Fc region, including hinge or aportion thereof, of a human immunoglobulin; and a moiety targeting thefusion protein to sites of calcification. Preferably, the Fc region isfrom human IgG1. Preferably, the moiety targeting the fusion protein tosites of calcification is Asp₁₀ (SEQ ID NO:18). More preferably, the Fcregion is from human IgG1 and the moiety targeting the fusion protein tosites of calcification is Asp₁₀ (SEQ ID NO:18).

Another preferred fusion protein comprises, from N-terminus toC-terminus, an NPP1 component comprising a portion of the cysteine-richdomain, the catalytic domain and the c-terminal region of human NPP1;optionally a linker (e.g., (Gly₄Ser)₃ (SEQ ID NO:16)); the Fc region,including hinge or a portion thereof, of a human immunoglobulin.Preferably, the Fc region is from human IgG1. In particular embodiments,the fusion protein has at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identityto SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. Preferredfusion protein of this type have the amino acid sequence of SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.

In particularly preferred aspects, a fusion protein of SEQ ID NO:3 isadministered in accordance with the methods described herein. In otherparticularly preferred aspect, a fusion protein of SEQ ID NO:4 isadministered in accordance with in the methods described herein. Inother particularly preferred aspect, a fusion protein of SEQ ID NO:9 isadministered in accordance with in the methods described herein. Inother particularly preferred aspect, a fusion protein of SEQ ID NO:10 isadministered in accordance with the methods described herein. In otherparticularly preferred aspect, a fusion protein of SEQ ID NO:11 isadministered in accordance with the methods described herein. In otherparticularly preferred aspect, a fusion protein of SEQ ID NO:12 isadministered in accordance with the methods described herein.

Fusion proteins of the present invention can be prepared using standardmethods, including recombinant techniques or chemical conjugation wellknown in the art. Techniques useful for isolating and characterizing thenucleic acids and proteins of the present invention are well known tothose of skill in the art and standard molecular biology and biochemicalmanuals can be consulted to select suitable protocols for use withoutundue experimentation. See, for example, Sambrook et al., 1989,“Molecular Cloning: A Laboratory Manual”, 2^(nd) ed., Cold SpringHarbor, the content of which is herein incorporated by reference in itsentirety.

The isolated recombinant human sNPP1, fragment, and fusion proteinsthereof, can be produced in any useful protein expression systemincluding, without limitation, cell culture (e.g., CHO cells, COS cells,HEK203), bacteria such as Escherichia coli (E. coli) and transgenicanimals, including, but no limited to, mammals and avians (e.g.,chickens, quail, duck and turkey). For expression, a construct thatencodes the sNPP1 and includes a suitable signal sequence (e.g, fromhuman Ig heavy chain, NPP2, NPP4, NPP7 or human serum albumin, forexample) in frame with the sequence of the sNPP1 and operably linked tosuitable expression control elements.

The sNPP1, including the fusion proteins, and physiologically acceptablesalt forms thereof are typically formulated into a pharmaceuticalcomposition for administration in accordance with the methods describedherein. Pharmaceutical compositions typically include a pharmaceuticallyacceptable carrier or excipient. Compositions comprising such carriers,including composite molecules, are formulated by well-known conventionalmethods (see, for example, Remington's Pharmaceutical Sciences, 14^(th)ed., Mack Publishing Co., Easton, Pa.), the entire teachings of whichare incorporated herein by reference. The carrier may comprise adiluent. In one embodiment, the pharmaceutical carrier can be a liquidand the fusion protein may be in the form of a solution. Thepharmaceutical carrier can be wax, fat, or alcohol. In anotherembodiment, the pharmaceutically acceptable carrier may be a solid inthe form of a powder, a lyophilized powder, or a tablet. In oneembodiment, the carrier may comprise a liposome or a microcapsule. Thepharmaceutical compositions can be in the form of a sterile lyophilizedpowder for injection upon reconstitution with a diluent. The diluent canbe water for injection, bacteriostatic water for injection, or sterilesaline. The lyophilized powder may be produced by freeze drying asolution of the fusion protein to produce the protein in dry form. As isknown in the art, the lyophilized protein generally has increasedstability and a longer shelf life than a liquid solution of the protein.

EXAMPLES

The present invention is further exemplified by the following examples.The examples are for illustrative purpose only and are not intended, norshould they be construed as limiting the invention in any manner.

Methods

Animals:

Six week old wildtype male C57B1/6J mice were used. The average weightof these mice ranged from 21-22 g. Mice were dosed with sNPP1-Fc [1.04mg/ml] or sNPP1-Fc-D10 [1.03 mg/ml] by subcutaneous (SC) or intravenous(IV) injection at a concentration of 5 mg/kg. Table 1.

TABLE 1 ID Drug/Route Time (h) 1 No treatment 0 2 No treatment 0 3sNPPI-Fc/IV 1 4 sNPPI-Fc/IV 1 5 sNPPI-FcD10/IV 1 6 sNPPI-FcD10/IV 1 7sNPPI-Fc/SC 4 8 sNPPI-Fc/SC 4 9 sNPPI-FcD10/SC 4 10 sNPPI-FcD10/SC 4

Two different strains of mice lacking NPP1 were used. Enpp1^(−/−) micewere previously described in Lomashvili, K. A. et al., Kidney Int 2014,85, 1351-1356. To accelerate arterial calcification, the diet wassupplemented with 1.5% phosphate (final phosphorus content: 2%) using amixture of NaH₂PO₄ and Na₂HPO₄ in proportions to yield a neutral pH aspreviously described. (O'Neill, W. C. et al., Kidney Int 2011, 79,512-517).

Chronic Kidney Disease (CKD) model: Wild-type sprague dawley rats wereused in CKD model studies. The rats were fed a diet containing0.25-0.75% adenine and high levels of phosphorus (0.75-0.9% phosphorusversus 0.4% in normal chow). The excess dietary adenine saturates thenormal adenine phosphoribosyltransferase salvage pathway and is insteadmetabolized to 2,8-dihydroxyadenine, which precipitates and formscrystals in the kidney tubules due to its low solubility. These crystalscause tubular injury, inflammation, obstruction, and fibrosis in thekidneys and lead to a phenotype consistent with human CKD. The resultingkidney damage and renal failure leads to impaired phosphate excretionresulting in abnormally high serum Pi levels and disordered mineralmetabolism, such as general calcification of soft tissues. The highlevel of phosphorus in the diet accelerates arterial calcification. Ratson the high adenine diet develop uremia, hyperphosphatemia, secondaryhyperparathyroidism, renal osteodystrophy, and vascular calcification.

Plasma Preparation:

Blood was collected by cardiac puncture and immediately mixed (9:1vol:vol blood to 110 mM citric acid solution). Serum collection resultsin release of excess pyrophosphate (PPi) from platelets, and EDTAinhibition of clotting may interfere with the assay. The tubes ofcitrated blood were nutated for several minutes and then spun at 2,000×gfor 10-15 min. The top layer of plasma was collected (100-300 μl) andapproximately 200 μl was added to a 10 kDa centricon. These tubes arethen spun at 12,000×g for 10 min to deproteinize the plasma. After thespin, the flow-through liquid was collected into a new tube. The plasmaand deproteinized samples are frozen at −20° C. until analysis.

Fluorometric PPi Assay:

This assay employs a fluorogenic PPi sensor that has its fluorescenceintensity proportionally dependent upon the PPi concentration. 10 kDafiltered samples (4 μl) was added to 46 μl of assay buffer. PPi sensorstock solution (200×) was diluted in assay buffer and 50 μl of this wasadded to the sample. After room temperature incubation for 20 min, thesolid black 96-well plate was read for fluorescence (Ex/Em=316/456 nm).

Assays:

NPP1 activity was measured as previously described. (Villa-Bellosta, R.et al., Am J Physiol Heart Circ Physiol 2011, 301, H61-H68). Briefly,plasma was added to 20 volumes of physiologic buffer containing 200 nMATP and 1.5 uCi [32P] ATP/ml for 10 minutes at 37° C. The reaction wasthen separated by thin-layer chromatography on polyethyleneiminecellulose and the amount of PPi produced was determined by densitometryof autoradiograms. Plasma PPi was measured as previously described(Lomashvili, K. A. et al., Kidney Int 2014, 85, 1351-1356), using plasmafreshly filtered through a 30 kD cut-off filter and an enzyme assaybased on the conversion of PPi and UDP-glucose to UTP andglucose-1-phosphate by UDPglucose pyrophosphorylase. All water used waspretreated with hydroxyapatite to remove contaminating PPi. Aorticcalcium was measured calorimetrically in HCl acid extracts of driedaortas as previously described. (Lomashvili, K. A. et al., Kidney Int2014, 85, 1351-1356). Calcium content was normalized to dry weight andfractional reductions in calcification were determined after subtractingthe calcium content of normal mouse aortas.

Blood Cell Fractionation:

To prepare leukocytes and platelets, freshly drawn, heparinized humanblood was centrifuged at 250 g for 15 minutes at room temperature. Theplasma was removed and centrifuged at 2200 g for 12 minutes to obtainplatelets. The pellet from the first centrifugation was re-suspended innormal saline to the original blood volume and 4 volumes of lysis buffer(155 mmol/L ammonium chloride; 10 mmol/L sodium bicarbonate; 0.1 mmol/LEDTA, pH 7.4) was added on ice for 5-10 minutes. This was repeated aftercentrifugation and removal of the supernatant, yielding purifiedleukocytes after a final centrifugation.

Statistical Analysis:

Continuous variables are expressed as means±standard errors withdifferences determined by Student's t-test. Aortic calcium content wasanalyzed after logarithmic transformation.

Example I

Background:

The experiment was conducted to determine whether there is an increasein PPi levels of wild-type mice that are dosed with variants of sNPP1.For this, 1 hour time point was selected for a single intravenousinjection therapy and 4 hour time point for single subcutaneousinjection therapy. The estimation of PPi levels was determined by theabcam PPi fluorometric assay.

Results:

The raw data from 1 min reads (9 total reads) were averaged andconverted to % of normal plasma (WT). Table 2

TABLE 2 Blank Blank Buffer Buffer WT1 WT2 IV Fc-1 IV Fc-2 IV D10-1 IVD10-2 sc Fc-1 sc Fc-2 sc D10-1 sc D10-2 1 0.4 0.4 32.6 31.1 36.2 40.237.9 48.0 51.0 40.6 47.0 45.9 46.3 47.3 2 0.4 0.4 31.5 30.1 36.3 40.837.2 46.7 50.8 39.0 46.5 44.6 46.7 47.3 3 0.4 0.4 31.5 31.1 35.5 40.837.0 45.7 51.0 38.8 46.2 44.1 46.0 46.2 4 0.4 0.4 31.1 31.3 35.5 40.437.0 46.0 49.5 38.8 46.6 45.3 45.6 46.2 5 0.4 0.4 31.2 29.9 35.5 39.735.4 45.7 50.3 38.6 46.3 43.7 46.4 46.4 6 0.3 0.4 31.0 29.8 35.4 40.236.0 44.7 50.9 39.0 45.7 44.2 44.4 44.8 7 0.4 0.4 30.7 31.2 34.2 39.635.1 45.5 50.9 38.6 45.8 43.5 45.5 45.7 8 0.4 0.4 32.0 29.4 34.9 40.835.5 45.4 50.4 37.7 45.0 43.6 46.1 44.5 9 0.4 0.3 31.0 29.6 34.3 38.935.6 45.3 51.3 37.1 45.7 43.4 44.8 45.1 ave 0.4 0.4 31.4 30.4 35.3 40.136.3 45.9 50.7 38.7 46.1 44.2 45.7 45.9

Intravenous or subcutaneous injection of sNPP1 protein variants (5mg/kg) in the wild-type mice shows an increase of PPi concentrationabove normal plasma levels as shown in FIG. 5 . FIG. 5 illustratespyrophosphate level in blood in wild-type mice after administration ofsNPP1-Fc or sNPP1-Fc-D10 intravenously (1 hour post injection) andsubcutaneously (4 hour post injection).

Example II

Enpp1(−/−) knock-out mice were treated subcutaneously with vehicle or 6mg/kg sNPP1-Fc-D10 every other day over a period of 21 days. Aorticcalcium levels are shown for males and females. FIG. 6 shows effectiveprevention of aortic calcification in Enpp1(−/−) mice with sNPP1-Fc-D10treatment.

Example III

Enpp1(−/−) knock-out mice was treated with 6 mg/kg sNPP1-Fc-D10intravenously to determine blood PPi and enzymatic activity levels. Asshown in FIG. 7 , plasma at time points of 0, 4, 24, 48, and 72 hourswere collected and analyzed for NPP1 activity (dashed) and PPi levels(solid). The wild-type PPi level was determined to be 2.18 μM (data notshown). The dashed lines from top to bottom show the PPi levels forwild-type, heterozygous Enpp1(+/−), and homozygous Enpp1(−/−) mice (Liet. al, 2013). The profiles for sNPP1-Fc were similar to those ofsNPP1-Fc-D10.

Example IV

Wild-type and Enpp1^(asj) mice were placed on a high phosphorus, lowmagnesium diet starting at birth. Vehicle or sNPP1-Fc (5 mg/kg) was dosesubcutaneously every other day starting at 14 days of age. Kaplan-Meiersurvival curves showed that >50% of asj mice died prior to 6 weeks, andall animals died by 9 weeks. In comparison, 50% of sNPP1-Fc treatedanimals survived past 7 week and are still living at 9 weeks. FIG. 8illustrates increased survival of Enpp1^(asj) homozygous male micetreated with 5 mg/kg sNPP1-Fc in comparison to vehicle treated mice.

Example V

Wild-type and Enpp1^(asj) mice were placed on a high phosphorus, lowmagnesium diet starting at birth and treated with vehicle or sNPP1-Fc (5mg/kg) subcutaneously every other day starting at 14 days of age todetermine growth rates. As shown in FIGS. 9A and 9B, percent body weightgain for wild-type (solid line) and Enpp1^(asj) (circles) mice wereplotted from two to nine weeks of age. FIGS. 9A and 9B illustratesincreased percent body weight gain of Enpp1^(asj) male mice treated with5 mg/kg sNPP1-Fc in comparison to vehicle treated mice. All Enpp1^(asj)animals were dead (open circle) in the vehicle group at nine weeks(upper panel). In comparison, five Enpp1^(asj) mice were alive (solidcircle) and five were dead (open circle) in the sNPP1-Fc treatment groupat the end of nine weeks. FIGS. 10A-10C illustrate pictures of wild-type(FIG. 10A, top), vehicle treated Enpp1^(asj) (FIG. 10B, middle) sNPP1-Fctreated (5mg/Kg) treated Enpp1^(asj) (FIG. 10C, bottom) mice.

Example VI

FGF-23 (Fibroblast growth factor 23), a biomarker for phosphatemetabolism, was measure in wild-type and Enpp1^(asj) male mice.Wild-type and Enpp1^(asj) mice were placed on a high phosphorus, lowmagnesium diet (TD.00442, Harlan) starting at birth. Vehicle orsNPP1-Fc-D10 (5 mg/kg) was dosed subcutaneously every other day startingat 18 days of age. All serum was collected 24 hours after dosing andanalyzed using a mouse FGF-23 ELISA kit (Kainos Laboratories Inc.,Tokyo, Japan). FGF-23 levels were measured at baseline (day 0), prior toinitiation of treatment and during the course of treatment inEnpp1^(+/+)-Vehicle (solid black), Enpp1^(asj/asj)-Vehicle (dottedblack), and Enpp1^(asj/asj)-sNPP1-Fc-D10 (solid grey) mice.

FGF-23 levels were elevated in Enpp1^(asj/asj) mice during the course ofdisease progression (by day 9 [27 days old]). However, theEnpp1^(asj/asj) mice treated with 5 mg/kg of sNPP1-Fc-D10 showed adecreased level of FGF-23 as compared to the vehicle treated group byday 17 of treatment. *, p<0.05 by one-way ANOVA or Student's t-test.FIG. 11 illustrates levels of fibroblast growth factor vehicle treatedEnpp1^(asj/asj) (middle) sNPP1-Fc treated (5 mg/Kg) treated Enpp1^(asj)(bottom) mice.

Example VII In Vitro and In Vivo Activity

The recombinant sNPP1-Fc-D10 fully hydrolyzed ATP to PPi in vitro withno hydrolysis of the PPi to orthophosphate as shown in FIG. 13A.

The enzyme activity in plasma is shown in FIG. 13B. Substantial activitywas present in the plasma of wild-type mice, with slightly more than onethird of the ATP converted to PPi in 10 minutes corresponding to anactivity of 7.6±1.0 nmol/h/ml. The remainder was converted toorthophosphate via nucleotide triphosphatases. Plasma from Enpp1^(−/−)mice was essentially devoid of NPP1, with the small amount of PPirepresenting PPi contaminating the [32P] ATP. Activity was markedlyincreased to 10.3±0.3 nmol/h/ml two hours after intravenous injection ofNPP1 (5 mg/kg) and this was accompanied by an increase in plasma PPifrom 0.07±0.02 to 1.00±0.14 uM, compared to a level of 2.39±0.37 uM inwild-type mice.

NPP1 activity was not detectable in aortas from either wild-type orEnpp1^(−/−) mice and did not increase after injection of NPP1 as shownin FIG. 13C. Activity was also not detected in liver after theadministration of recombinant NPP1.

The time course of plasma NPP1 activity and PPi concentration aftersubcutaneous injection of 5 mg/kg into Enpp1^(−/−) mice is shown in FIG.14 . NPP1 activity and PPi concentration peaked 12 hours after injectionat levels that were 195% and 41% respectively of those in wild-typelittermates. The levels decreased rapidly and were essentiallyundetectable after 24 hours.

Subcutaneous injection of sNPP1-Fc-D10 (5 mg/kg) shows a correlationbetween the plasma PPi levels and plasma NPP1 activity as shown in FIG.15 . The correlation of plasma PPi with plasma NPP1 suggested that thePPi was generated in the circulation. This was examined by incubatingfresh human blood with recombinant NPP1 and then measuring PPi in theplasma. Human blood was used because of the limited amount of bloodobtainable from mice. The amount of NPP1 added to the blood wascalculated so as to yield levels similar to those achieved afterinjection in mice.

FIG. 16A illustrates that administration of recombinant NPP1 increasedplasma PPi when added to whole blood for 2 hours but not when added toplasma alone, indicating a cellular requirement. To examine the role oferythrocytes versus other cells, blood was centrifuged and plasma wasremoved either with or without the buffy coat remaining. HEPES-bufferedsaline was then added to restore the original hematocrit. As shown inFIG. 16B, production only occurred when the buffy coat was retained,indicating a requirement for leukocytes or platelets but noterythrocytes. Incubation of isolated leukocytes or platelets inHEPES-buffered saline indicated that both either released or producedPPi but that synthesis in response to exogenous NPP1 occurred only withleukocytes as shown in FIG. 16C.

Example VIII Therapeutic Models

A. NPP1 Deficiency

Enpp1^(−/−) mice aged were placed on a high phosphate diet and treatedwith vehicle or sNPP1-Fc-D10 (6 mg/kg) subcutaneously every other day asshown in FIG. 17 to determine the effect of recombinant NPP1 on arterialcalcification. Each treated mouse was paired with a mouse of the samegender and similar age that received the same volume of vehicle alone.After 18 days, the mean aortic calcium content was 61±30 nmol/mg in thevehicle-treated mice and 8.8±1.0 nmol/mg in the mice treated withrecombinant NPP1 (p=0.016). The content in wild-type littermates was6.3±3.4 nmol/mg (n=16). Content was elevated (two standard deviationsabove wild-type littermates) in 6 of 8 control aortas (80±37 nmol/mg)and in only one treated aorta (15 nmol/mg). Within the pairs in whichcalcification was present in control aortas, this represented a 91±2%decrease in calcification.

To determine whether there is any accumulation of NPP1 after multipleinjections over time, plasma NPP1 activity and PPi, measured atsacrifice (24 hours after injection), and were both undetectable. In aseparate set of Enpp1^(−/−) mice, aortic NPP1 activity was undetectableafter 3 injections of recombinant NPP1 every other day.

B. Chronic Kidney Disease

This example discloses the efficacy of sNPP1-Fc-D10 in treating chronickidney disease (CKD) in uremic rat models. To determine the effect ofrecombinant NPP1 on arterial calcification in uremic rats with renalfailure, the uremic rats were fed a high adenine diet and injectedsubcutaneously with control or sNPP1-Fc-D10 (5 mg/kg), 5 dose per weekas illustrated in FIG. 18 . After 21 days of treatment, the mean aorticcalcium content was 25.7±4.9 nmol/mg in the control-treated rat and7.0±1.0 nmol/mg in the rat treated with recombinant NPP1 (p=0.0068). Thenormal aortic calcium content was 5 nmol/mg.

Examples VII and VIII demonstrate the activity of sNPP1 and effectiveuse of sNPP1 in models of ectonucleotide pyrophosphate pyrophosphorylasedeficiency and chronic kidney disease. These examples show that atransient increase in PPi is sufficient for an effective therapy ofvascular calcification and NPP1 deficiency.

EQUIVALENTS

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method for reducing vascular calcificationtreating a subject having Pseudoxanthoma elasticum (PXE), comprisingadministering to a the subject with below normal plasma pyrophosphate(PPi) or above normal serum phosphate (Pi) two or more doses of aneffective amount of soluble ectonucleotide pyrophosphatasephosphodiesterase (sNPP1), fragment or fusion protein thereof, whereineach dose contains an amount of sNPP1 that is sufficient to achieve atransient increase in plasma PPi in the subject, the transient increasein plasma PPi characterized by a peak plasma PPi level that is at leastabout 40% of the normal plasma PPi level and a return to base-lineplasma PPi level within about 48 hours after administration of the dose;wherein a) the time period between doses is at least 2 days; b) thenormal level of plasma PPi is 2.63±0.47 microMolar; c) the normal levelof plasma Pi is 1.5±0.5 milliMolar; and d) wherein sNPP1 haspyrophosphatase activity, phosphodiesterase activity, or pyrophosphataseand phosphodiesterase activity, with the proviso that when the sNPP1 isa fusion protein comprising an NPP1 component and one or more fusionpartners, each fusion partner is located C-terminally to the NPP1component.
 2. The method of claim 1, wherein the transient increase inplasma PPi is maintained for at least about 4 hours.
 3. The method ofclaim 1, wherein the vascular subject has calcification is arterialcalcification of soft tissue.
 4. The method of claim 1, wherein thevascular calcification is intimal calcification subject hascalcification of the skin and/or eye(s).
 5. The method of claim 1,wherein said subject has NPP1 deficiency.
 6. The method of claim 1,wherein the subject has chronic kidney disease (CKD) or end-stage renaldisease (ESRD).
 7. The method of claim 1, wherein the subject hasgeneralized arterial calcification of infancy (GACI).
 8. The method ofclaim 1, wherein the subject has a cardiovascular disorder, diabetesmellitus II, atherosclerosis, or Pseudoxanthoma elasticum (PXE).
 9. Themethod of claim 1, wherein the levels of plasma pyrophosphate (PPi) inthe subject before treatment is at least about 40% lower than that ofthe normal plasma PPi levels.
 10. The method of claim 1, wherein thesubject is human.
 11. The method of claim 1, wherein each dose containssaid effective amount comprises about 1.0 0.1 mg/kg to about 10.0 2.0mg/kg sNPP1.
 12. The method of claim 1, wherein time period between saidsNPP1 doses is at least 3 days.
 13. The method of claim 1, wherein theadministration is intravenous, subcutaneous, or intraperitoneal.
 14. Themethod of claim 1, wherein the sNPP1 comprises an isolated recombinanthuman sNPP1.
 15. The method of claim 1, wherein the sNPP1 is a fusionprotein comprising a) an NPP1 component that lacks the N-terminalcytosolic and transmembrane domains, and b) a fusion partner locatedC-terminally to the NPP1 component.
 16. The method of claim 15, whereinthe fusion protein further comprises a targeting moiety.
 17. The methodof claim 1, wherein the sNPP1 is SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.
 18. The method of claim 1,wherein the subject has elevated inorganic phosphate and a ratio of PPito Pi that is at least 10% higher or lower than normal.
 19. The methodof claim 15, wherein the fusion partner comprises the Fc region of animmunoglobulin.
 20. The method of claim 15, wherein the fusion proteinfurther comprises a linker, a peptide that targets the fusion protein tosites of calcification, or a linker and a peptide that targets thefusion protein to sites of calcification.
 21. The method of claim 11,where said effective amount comprises 0.5 mg/kg sNPP1.
 22. The method ofclaim 11, wherein said effective amount comprises 1.0 mg/kg sNPP1. 23.The method of claim 11, wherein said effective amount comprises 5.0mg/kg sNPP1.
 24. The method of claim 11, wherein said effective amountcomprises 10 mg/kg sNPP1.
 25. The method of claim 1, wherein saidadministration is weekly.
 26. The method of claim 1, wherein saidadministration is bi-weekly.
 27. The method of claim 1, wherein saidadministration is monthly.
 28. The method of claim 13, wherein saidadministration is intravenous.
 29. The method of claim 13, wherein saidadministration is subcutaneous.